This is a proposal to test the feasibility of using triplex-forming oligonucleotides to correct the sickle cell disease (SCD) mutation in human CD34+ cells. SCD is one of the most common human genetic diseases and is the result of one specific inherited mutation in the 2-globin gene. Hydroxyurea is currently the only FDA-approved treatment for SCD. Both supportive and preventive measures remain the mainstay of treatment for this disease. Allogeneic hematopoietic stem cell transplantation is curative, although such a treatment typically requires an HLA-matched donor, is associated with significant morbidity and is very expensive. However, genetic modification of autologous CD34+ cells is becoming a viable therapeutic modality. Recent studies suggest that this technique can be used to treat hemoglobin disorders. Current methods for correcting mutations in CD34+ cells require non-directed insertion of viral vectors. This method is expensive, has low success rates and carries a high risk of induced tumorigenesis. Some non-viral methods have been developed for directly correcting the genes of interest in CD34+ cells. However, these current approaches are expensive and have a low rate of success. To address the above weaknesses in current curative methods, we propose to test the feasibility of using our triplex-forming oligonucleotide approach. Our procedure has been shown to stimulate recombination in mammalian cells by the ability of triple helices to provoke DNA repair and, thus, sensitize the target site to recombination. This technology constitutes minimally invasive gene repair, as gene modification occurs in situ via use of the cell's own DNA repair machinery, without the need for viral vectors. Helix Therapeutics was formed to commercialize this technology for treating common human diseases, including SCD. Our approach promises to be safe and inexpensive. In the proposed studies, we will determine an effective combination of triplex and donor DNA molecules, as well as an effective cellular delivery method for our gene targeting molecules. Our primary proof of principle will be to demonstrate that this method can induce directed mutations of the 2-globin gene at a rate of 10% or higher, which is the expected level required to become clinically effective. We will carry out this proof of principle test in human CD34+ cells and evaluate whether the cells are able to engraft and properly differentiate in a mouse model of stem cell transplantation. These experiments will constitute a proof-of-concept study. Additional improvements to the methods and other studies necessary for filing an IND for clinical testing will take place in a Phase II application. PUBLIC HEALTH RELEVANCE: Sickle cell disease (SCD), also called sickle cell anemia, is among the most common human genetic disorders and is the result of one specific inherited mutation in the 2-globin gene, which is involved in the synthesis of hemoglobin. Hemoglobin is the protein in red blood cells that carries oxygen. Helix Therapeutics is proposing to develop a therapeutic gene targeting agent to correct, in human hematopoietic stem cells, the mutation responsible for causing SCD and, thus, cure the disease permanently.